This application describes a two-year research scholar development program analyzing Vibrio cholerae quorum sensing in vivo with the long term goal of establishing an outstanding, well funded research program in the cell-cell communication in bacterial pathogens. Many important bacterial functions, including virulence in several clinically important human pathogens, are regulated by the exchange of chemical signals, permitting a population of bacteria to coordinate their responses to environmental challenges or opportunities. Recent studies show that quorum sensing plays an important role in the pathogenesis of V. cholerae, the causative agent of cholera. The first specific aim of this application is to use a combination of biochemical, genetic, and genomic methods to analyze quorum sensing-mediated control of V. cholerae pathogenesis. I will develop a genetic tool by introduce a second parallel reporter system to the established RIVET approach to study the temporal and spatial expression of V. cholerae virulence genes during infection. Correlations between quorum signal accumulation and virulence gene expression will be investigated in an infant mouse model. High throughput screening of chemical libraries will be performed to identify organic molecules that have Vibrio quorum-signal activites. Next, novel in vivo quorum sensing-regulated genes will be identified using a modified differential fluorescent induction approach, and their cellular functions will be analyzed. In addition, the roles that V. cholerae biofilms play in infection will be investigated. Host-pathogen interactions required to stimulate biofilm formation in V. cholerae will be studied, and the role of biofilm development in determing the infectivity of V. cholerae isolated from patients will be examined. The basic underlying goal of this proposal is apply the study of V. cholerae quorum sensing in vivo to the discovery of potential novel treatments for cholera disease.